Lesson 10: Enzyme Inhibitors, Cofactors and the Effects of pH, Temperature and Concentration
Enzyme Inhibitors, Cofactors and the Effects of pH, Temperature and Concentration
Enzymes are tightly regulated biological machines. Their activity can be modified by inhibitors, enhanced by cofactors, and affected by environmental conditions such as pH, temperature and substrate concentration. This lesson covers the OCR A-Level Biology A specification points 2.1.4 (c)–(g) — the effects of pH, temperature, enzyme and substrate concentration on the rate of enzyme-catalysed reactions; the roles of cofactors, coenzymes and prosthetic groups; and the effects of competitive and non-competitive inhibitors, including end-product inhibition.
This is the final lesson in the nucleic acids and enzymes topic. Together with Lesson 9, it completes the OCR specification content for Chapter 4 (in most OCR textbooks).
1. Factors Affecting Enzyme Activity
Enzyme activity (measured as the rate of reaction, usually the rate at which substrate is used up or product is formed) is affected by several factors:
Temperature
pH
Enzyme concentration
Substrate concentration
Presence of inhibitors
Presence of cofactors, coenzymes or prosthetic groups
OCR expects you to be able to describe, explain and draw graphs of each of these effects.
2. Effect of Temperature
As temperature increases, molecules gain kinetic energy. Enzyme and substrate molecules collide more frequently and with more energy, so the number of successful enzyme–substrate complexes formed per second increases. The rate rises.
Each enzyme has an optimum temperature (around 37 °C for most human enzymes, much higher for enzymes in thermophilic bacteria).
Above the optimum, the increased vibration of atoms in the enzyme begins to break the weak bonds (hydrogen bonds and ionic bonds) that hold the tertiary structure together. The active site changes shape, and eventually the enzyme denatures and loses all activity.
Denaturation is usually permanent — cooling a denatured enzyme does not restore its function.
Q10
The temperature coefficient (Q10) is the factor by which a reaction rate increases for a 10 °C rise in temperature. For most enzymes below the optimum, Q10 ≈ 2 (the rate roughly doubles for every 10 °C rise).
3. Effect of pH
Each enzyme has an optimum pH, at which the active site has its ideal 3D shape and charge distribution.
Either side of the optimum, the rate decreases.
Why? pH is a measure of H⁺ concentration. Changes in pH alter the charges on acidic (–COOH) and basic (–NH₂) side chains in the enzyme, disrupting the hydrogen bonds and ionic bonds that maintain the tertiary structure. The active site changes shape; the substrate can no longer bind properly.
Extreme changes cause denaturation, again usually permanent.
Examples of Optimum pH
Enzyme
Location
Optimum pH
Pepsin
Stomach
~2
Salivary amylase
Mouth
~7
Trypsin
Small intestine
~8
Exam Tip: Always refer to specific bonds (hydrogen and ionic bonds) in the tertiary structure when explaining pH effects. Just saying "the enzyme stops working" does not score marks.
4. Effect of Enzyme Concentration
Provided substrate is in excess, increasing enzyme concentration increases the rate of reaction in direct proportion — more active sites are available, so more ES complexes form per second.
Eventually the rate levels off because the substrate concentration becomes limiting — every substrate molecule can find an active site, and adding more enzyme makes no difference.
5. Effect of Substrate Concentration
At low substrate concentrations, most active sites are empty — adding more substrate gives a proportional increase in rate.
As substrate concentration increases, the rate levels off because all the active sites are occupied at any given moment. The enzyme is working at maximum rate (Vmax).
Adding more substrate beyond this point has no further effect — only adding more enzyme can increase the rate.
6. Cofactors, Coenzymes and Prosthetic Groups
Some enzymes are fully active on their own. Many others need helper molecules to work properly. These helpers are called cofactors (a general term) and come in several forms.
Key Definition — Cofactor: A non-protein substance that is needed for the proper functioning of an enzyme. Cofactors may be inorganic ions, organic coenzymes or prosthetic groups.
6.1 Inorganic Ion Cofactors
These are simple metal ions that bind to the enzyme or substrate to enable catalysis.
Cl⁻ is a cofactor for amylase.
Mg²⁺ is a cofactor for many ATP-using enzymes, including DNA polymerase and kinases.
6.2 Coenzymes
Coenzymes are organic molecules that bind temporarily to an enzyme (or, more often, shuttle between enzymes) to transfer a chemical group. They are not permanently attached.
Most coenzymes are derived from vitamins — which is why vitamin deficiencies cause metabolic problems.
Coenzyme
Role
Vitamin origin
NAD (nicotinamide adenine dinucleotide)
Carries hydrogen (reducing power) in respiration
Niacin (B3)
FAD (flavin adenine dinucleotide)
Carries hydrogen in respiration
Riboflavin (B2)
Coenzyme A (CoA)
Carries acetyl groups in respiration
Pantothenic acid (B5)
Exam Tip: NAD, FAD and Coenzyme A are all coenzymes that you need to know for OCR — they will reappear in the Year 2 topic on respiration.
6.3 Prosthetic Groups
Prosthetic groups are non-protein components that are permanently, covalently bound to an enzyme and are essential for its function.
Prosthetic group
Enzyme
Role
Fe²⁺ (haem)
Catalase
Needed to break down hydrogen peroxide
Zn²⁺
Carbonic anhydrase
Needed to interconvert CO₂ and HCO₃⁻ in red blood cells
Both catalase and carbonic anhydrase are examples you should learn for OCR-style questions. Carbonic anhydrase is one of the fastest enzymes known — each molecule can hydrate around 10⁶ CO₂ molecules per second.
Exam Tip: A common mistake is to say that prosthetic groups are the same as coenzymes. They are not. Prosthetic groups are permanently attached; coenzymes shuttle between enzymes.
7. Enzyme Inhibitors
Inhibitors are molecules that reduce the rate of an enzyme-catalysed reaction. They fall into several categories.
7.1 Competitive Inhibitors
A competitive inhibitor has a shape similar to the substrate and binds to the active site, blocking substrate binding.
Reversible — the inhibitor binds and releases repeatedly.
Its effect depends on the ratio of inhibitor to substrate. Increasing the substrate concentration can outcompete the inhibitor and restore normal activity (or nearly so).
Example: malonate is a competitive inhibitor of succinate dehydrogenase in the Krebs cycle. It resembles succinate.
Example: statin drugs are competitive inhibitors of HMG-CoA reductase in cholesterol synthesis.
7.2 Non-Competitive Inhibitors
A non-competitive inhibitor binds to a site on the enzyme other than the active site — called an allosteric site. Binding causes the tertiary structure of the enzyme to change, distorting the active site so that the substrate can no longer bind (or can no longer undergo the catalysed reaction).
Increasing substrate concentration does not overcome non-competitive inhibition, because the inhibitor is not competing for the same site.
Example: cyanide non-competitively inhibits cytochrome c oxidase in respiration.
Example: many heavy metal ions (Hg²⁺, Ag⁺) are non-competitive inhibitors of many enzymes.
7.3 Reversible vs Irreversible Inhibitors
Reversible inhibitors bind temporarily (via weak interactions) and dissociate. Their effects can be reversed by removing the inhibitor or adding more substrate.
Irreversible inhibitors bind by covalent bonds and remain permanently attached — enzyme activity is lost until new enzyme is synthesised. Nerve agents and organophosphate pesticides work this way on acetylcholinesterase.
7.4 Summary Graph — Effect on Rate vs [Substrate]
Key point: competitive inhibition can be overcome by raising the substrate concentration — non-competitive inhibition cannot.
8. End-Product Inhibition
End-product inhibition is a form of metabolic regulation in which the product of a metabolic pathway inhibits an earlier enzyme in the pathway, switching off further synthesis once enough product is present. It is a type of negative feedback.
graph LR
A[Substrate A] -- Enzyme 1 --> B[Intermediate B]
B -- Enzyme 2 --> C[Intermediate C]
C -- Enzyme 3 --> D[Product D]
D -- inhibits --> A_to_B[Enzyme 1]
Classic example: the amino acid isoleucine inhibits threonine deaminase, the first enzyme in its own biosynthesis pathway. When isoleucine levels rise, synthesis is switched off; when they fall, synthesis resumes.
Why is end-product inhibition important?
It prevents waste — the cell does not make more of a product than it needs.
It allows the cell to respond quickly to changes in demand.
It keeps metabolic pathways in balance.
End-product inhibitors are typically non-competitive (allosteric), binding to a site different from the substrate. This allows the regulation to be independent of substrate concentration.
9. Common Exam Mistakes
Saying that increasing temperature always increases the rate. It increases the rate up to the optimum; above that, the enzyme denatures and the rate falls.
Writing "denatured" without explaining that hydrogen and ionic bonds in the tertiary structure are broken and the active site changes shape.
Confusing coenzymes and prosthetic groups. Coenzymes shuttle between enzymes; prosthetic groups are permanently bound.
Saying a competitive inhibitor "changes the shape of the active site". It does not — it physically blocks the active site by binding there. Non-competitive inhibitors change the shape of the active site.
Saying high substrate concentration can "overcome" any type of inhibition. Only competitive inhibition can be outcompeted.
Forgetting that end-product inhibition is usually a form of non-competitive inhibition.
Writing that enzyme concentration always limits the rate. If substrate is limiting, adding more enzyme will not increase the rate.